CD task2 protocol

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Sequence

The native ASPA sequence (UniProt: P45381):

>hsa:443 ASPA, ACY2, ASP; aspartoacylase; K01437 aspartoacylase [EC:3.5.1.15] (A)
MTSCHIAEEHIQKVAIFGGTHGNELTGVFLVKHWLENGAEIQRTGLEVKPFITNPRAVKK
CTRYIDCDLNRIFDLENLGKKMSEDLPYEVRRAQEINHLFGPKDSEDSYDIIFDLHNTTS
NMGCTLILEDSRNNFLIQMFHYIKTSLAPLPCYVYLIEHPSLKYATTRSIAKYPVGIEVG
PQPQGVLRADILDQMRKMIKHALDFIHHFNEGKEFPPCAIEVYKIIEKVDYPRDENGEIA
AIIHPNLQDQDWKPLHPGDPMFLTLDGKTIPLGGDCTVYPVFVNEAAYYEKKEAFAKTTK
LTLNAKSIRCCLH

GO term enrichment

<source lang="java"> for(int i = 0; i< seq_id.length; i++ ){

System.out.println(seq_id[i]);

// URL for annotations from QuickGO for one protein URL u=new URL("http://www.ebi.ac.uk/QuickGO/GAnnotation?protein="+seq_id[i]+"&format=tsv"); // Connect HttpURLConnection urlConnection = (HttpURLConnection) u.openConnection(); // Get data BufferedReader rd=new BufferedReader(new InputStreamReader(urlConnection.getInputStream())); List<String> columns=Arrays.asList(rd.readLine().split("\t")); int idIndex=columns.indexOf("GO ID"); int nameIndex=columns.indexOf("GO Name"); String line; if(rd.ready()) count_go_prots++; Set<String> names = new HashSet<String>(); while ((line=rd.readLine())!=null) { // Split them into fields String[] fields=line.split("\t");

if(!names.contains(fields[nameIndex])){ names.add(fields[nameIndex]); if(this.go_ids.containsKey(fields[idIndex])) { int count = Integer.parseInt(this.go_ids.get(fields[idIndex])[1]); this.go_ids.put(fields[idIndex], new String[]{fields[nameIndex], String.valueOf(count+1)}); } else{ this.go_ids.put(fields[idIndex], new String[]{fields[nameIndex],"1"}); } } }

// close input when finished rd.close(); } </source>

BlastP

We ran BlastP on student machines with the big_80 as a reference database.

<source lang="bash"> blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o blastp_p45381_wt_big80.out </source>

default E-Value 10 - GO Term Enrichment (hit more than once)

#hits   GO term
185	 metabolic process
184	 hydrolase activity, acting on ester bonds
133	 hydrolase activity
125	 metal ion binding
88	 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
60	 aspartoacylase activity
44	 zinc ion binding
24	 arginine metabolic process
24	 arginine catabolic process to glutamate
23	 succinylglutamate desuccinylase activity
8	 cytoplasm
5	 arginine catabolic process to succinate
4	 apical plasma membrane
4	 aminoacylase activity
4	 membrane
3	 plasma membrane
3	 identical protein binding
2	 nucleus
2	 intracellular
2	 exonuclease activity
2	 nucleotide binding
2	 nucleic acid binding
2	 oxidoreductase activity
2	 oxidation-reduction process

E-Value 10e-10 - GO Term Enrichment (hit more than once)

#hits   GO term
94	 hydrolase activity, acting on ester bonds
94	 metabolic process
88	 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
66	 hydrolase activity
62	 metal ion binding
58	 aspartoacylase activity
19	 zinc ion binding
8	 cytoplasm
4	 apical plasma membrane
4	 aminoacylase activity
3	 plasma membrane
3	 identical protein binding
3	 membrane
2	 nucleus

PsiBlast

PSIBlast was used in the same fashion as BLAST, with the big_80 as the background database.

2 iterations and default E-Value 0.002 <source lang="bash"> blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_p45381_wt_big80.out -j 2 -v 700</source>


2 iterations, more strict E-value cutoff of 10E-10 <source lang="bash"> blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_h10e10_p45381_wt_big80.out -j 2 -h 10e-10 </source>

GO Term Enrichment (all terms represented more than once)

766	hydrolase activity, acting on ester bonds
665	metabolic process
507	hydrolase activity
488	metal ion binding
294	zinc ion binding
234	arginine catabolic process to glutamate
176	hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
158	aspartoacylase activity
132	arginine metabolic process
132	succinylglutamate desuccinylase activity
30	cytoplasm
24	arginine catabolic process to succinate
23	proteolysis
22	metallocarboxypeptidase activity
12	apical plasma membrane
9	carboxypeptidase activity
8	nucleus
8	aminoacylase activity
8	membrane
6	identical protein binding
6	oxidation-reduction process
6	plasma membrane
5	oxidoreductase activity
2	malate synthase activity
2	glyoxylate cycle
2 	exonuclease activity
2	nucleotide binding
2	acetate metabolic process
2	arginine catabolic process
2	cell adhesion
2	virus-host interaction
2	integral to membrane
2	transferase activity 



10 iterations, default Evalue 0.002 <source lang="bash"> blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_p45381_wt_big80.out -j 10 </source>


10 iterations, E-value cutoff 10E-10 <source lang="bash"> blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_h10e10_p45381_wt_big80.out -j 10 -h 10e-10 </source>

HHBlits

Run HHBlits on student machines with Uniprot20 database.

  • 2 iterations
 <source lang="bash">  hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -o hhblits_p45381_def.out </source>
  • 8 iterations

<source lang="bash"> hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -n 8 -z 1000 -v 1000-o hhblits_p45381_n10.out </source>

  • 2 iterations, -e 10e-10
 <source lang="bash">  hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -e 10e-10 -o hhblits_p45381_def.out </source>
  • 8 iterations, -e 10e-10

<source lang="bash"> hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -n 8 -e 10e-10 -z 1000 -v 1000 -o hhblits_p45381_n10.out </source>